Summary: Retroviral vectors have been used in clinical gene therapy trials to effect permanent cure of inborn errors of metabolism, such as ADA-SCID, because these vectors can integrate into the host genome to provide permanent transgene expression in the targeted cells. Current retroviral vectors used in clinical trials lack a selectable marker (neo) gene, to avoid immune responses to transfected cells. As a result, selection of transfected packaging cell lines has become an extremely labor intensive process involving the screening of thousands of clones by RNA analyses. We first addressed the task of facilitating selection of transfected packaging cell lines by adopting the strategy of using a selectable marker, but placing it outside of the inter-LTR regions of the retroviral construct. Thus, the selection marker is active in the packaging cells, but is not incorporated into the retroviral transcript produced by the packaging cell lines. We created a novel viral construct (MPSV-GFP)in which a neo gene selection marker was directly cloned into the plasmid backbone of MPSV-GFP, yielding (MPSV-GFP)-neo. We have demonstrated that this is an effective strategy in that within three weeks of transfection, 45% of packaging cells transfected with (MPSV-GFP)-neo and raised on a high concentration of G418, expressed the construct, compared to only 8% of packaging cells that were co-transfected with separate MPSV-GFP and neo constructs and produced a higher (2-3 fold) viral titre. We have further demonstrated that the selection marker is not present in target cells transfected with retrovirus produced by the(MPSV-GFP)-neo packaging cell lines, indicating that tandem repeats were not incorporated in the retroviral transcript. An invention report is being pursued by patent attorneys. The second project, in which we attempted to boost expression of a retroviral transgene in lymphocytes has revealed that the murine 3' light chain enhancer (mE3')has locus control region (LCR) activity in both reducing variability of expression and enhancing expression in both T cell lines and primary T cells. Interestingly, it failed to do same for B cell tumor lines. In vivo expression patterns mediated by the mE3' are being pursued. 1. Lai Xu 1, Kazuhide Tsuji1, Howard Mostowski, Makoto Otsu, Fabio Candotti and Amy Rosenberg. A convenient method fore generation of viral producing clones by placement of a positive selectable marker outside of the vector backbone of a simplified retroviral vector. Submitted 2. Lai Xu, Kazuhide Tsuji, Howard Mostowski, Fabio Candotti and Amy Rosenberg Evidence that the Mouse Kappa Light Chain 3' Enhancer Gene Confers Position-Independent Transgene Expression of a Simplified Retroviral Vector in T Lineage Cells. In preparation.